The Study On The Role Of TIPE3 In Promoting The Growth And Metastasis Of Colorectal Cancer Through EphA2 And Its Mechanism

Objective:To elucidate the role and mechanism of tumor necrosis factor-alpha-induced protein 8-like 3(TIPE3)in promoting the growth and metastasis of colorectal cancer through erythropoietin-producing human hepatocelluar A 2(EphA2).Methods:(1)The total proteins of human normal intestinal mucosal epithelial cells NCM460(for control)and colorectal cancer(CRC)cells HT29,RKO,LOVO,SW480 and SW620 were extracted,and the expression level of TIPE3 in CRC cells was detected by Western Blot.(2)The TIPE3-overexpression lentivirus plasmid(already constructed by the research group)and TIPE3-knockout lentivirus plasmid were constructed.These plasmids were packaged into TIPE3-overexpression lentivirus and TIPE3-knockout lentivirus,respectively.And these lentivirus were measured for titer.(3)The above-mentioned virus were used to infect colorectal cancer cells SW480 to construct TIPE3 stable overexpression cell lines and TIPE3 stable knockout cell lines,and the effects of TIPE3 overexpression and knockout on the growth,proliferation,migration and invasion of CRC cells were studied through CCK8,plate cloning,wound healing,Transwell migration,invasion and other cell function experiments.(4)The effect of TIPE3 on c-Myc and EphA2 in TIPE3 overexpressed or knocked out cells were detected by Western Blot,and the mRNA level of EphA2 in cells was detected by RT-qPCR.The expression level of EphA2 in SW480-TIPE3 cells was further detected by EphA2 flow antibody staining.(5)C-Myc siRNA and its control were transfected into SW480-TIPE3 cells,to detect its transfection efficiency and investigate the effect of c-Myc knockdown on EphA2 in cells.(6)The expression trend of EphA2 in CRC cells was analyzed by EphA2 flow antibody staining and CCLE database.(7)EphA2 siRNA and its control were transfected into SW480-TIPE3 cells,to detect its transfection efficiency and investigate whether knock down EphA2 could weaken the promoting effect of TIPE3 on colorectal cancer.Results:(1)TIPE3 was highly expressed in human CRC cells compared with human normal intestinal mucosa epithelial cell NCM460.(2)TIPE3-overexpression lentivirus and TIPE3-knockout lentivirus were successfully established.(3)TIPE3 overexpression promoted the growth,migration and invasion of CRC cells SW480 in vitro,TIPE3 knockout inhibited the growth,migration and invasion of CRC cells SW480 in vitro.(4)Overexpression of TIPE3 significantly increased the expression of c-Myc and EphA2 in the cells,while the expression of c-Myc and EphA2 in the cells decreased with the elimination of TIPE3.(5)C-Myc in SW480-TIPE3 was successfully interfered.Compared with control siRNA interference in SW480-TIPE3 control cells,EphA2 in SW480-TIPE3 cells was down-regulated by c-Myc siRNA interference.(6)The results of flow antibody detection of EphA2 expression level in CRC cells were basically consistent with the results of CCLE database.(7)EphA2 in SW480-TIPE3 was successfully interfered with.Compared with control siRNA interference with SW480-TIPE3 control cells,the growth and transfer of EphA2 siRNA interference with SW480-TIPE3 cells were significantly inhibited.Conclusions:TIPE3 is highly expressed in human colorectal cancer cells and promotes the growth and metastasis of CRC cells.The up-regulation of EphA2 in cells induced by TIPE3 through c-Myc is a crucial link in the process of promoting the growth and metastasis of CRC cells mediated by TIPE3.